Current Issues in Molecular Biology (Mar 2023)

Development of Digital Droplet PCR Targeting the Influenza H3N2 Oseltamivir-Resistant E119V Mutation and Its Performance through the Use of Reverse Genetics Mutants

  • Laura A. E. Van Poelvoorde,
  • François E. Dufrasne,
  • Steven Van Gucht,
  • Xavier Saelens,
  • Nancy H. C. Roosens

DOI
https://doi.org/10.3390/cimb45030165
Journal volume & issue
Vol. 45, no. 3
pp. 2521 – 2532

Abstract

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The monitoring of antiviral-resistant influenza virus strains is important for public health given the availability and use of neuraminidase inhibitors and other antivirals to treat infected patients. Naturally occurring oseltamivir-resistant seasonal H3N2 influenza virus strains often carry a glutamate-to-valine substitution at position 119 in the neuraminidase (E119V-NA). Early detection of resistant influenza viruses is important for patient management and for the rapid containment of antiviral resistance. The neuraminidase inhibition assay allows the phenotypical identification of resistant strains; however, this test often has limited sensitivity with high variability depending on the virus strain, drugs and assays. Once a mutation such as E119V-NA is known, highly sensitive PCR-based genotypic assays can be used to identify the prevalence of such mutant influenza viruses in clinical samples. In this study, based on an existing reverse transcriptase real-time PCR (RT-qPCR) assay, we developed a reverse transcriptase droplet digital PCR assay (RT-ddPCR) to detect and quantify the frequency of the E119V-NA mutation. Furthermore, reverse genetics viruses carrying this mutation were created to test the performance of the RT-ddPCR assay and compare it to the standard phenotypic NA assay. We also discuss the advantage of using an RT-ddPCR instead of qPCR method in the context of viral diagnostics and surveillance.

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